Cilia serve as sensory devices on most eukaryotic cell surfaces and play essential roles in organogenesis and tissue pattern formation during development. Ciliary assembly via intraflagellar transport (IFT) and sensory transduction capabilities are highly conserved in all ciliated organisms. With rapid advancements in the positional cloning of human disease genes in the past decade, a wide variety of disorders, such as autosomal dominant polycystic kidney disease (ADPKD), Joubert syndrome (JBST), Bardet-Biedl syndrome (BBS), nephronophthisis (NPHP), Meckel-Gruber syndrome (MKS), and autosomal recessive polycystic kidney disease (ARPKD), have been characterized molecularly as ciliopathies. The establishment and maintenance of ciliary function are clearly essential for the well-being of an organism. Consistent with the ubiquitous presence of cilia, many ciliopathies occur as syndromic disorders that affect multiple organs, including the kidneys, liver limbs, eyes, central nervous system (CNS), and fat storage tissue. Despite the physiological and clinical relevance of cilia, the core machinery that regulates cilia biogenesis and function as well as the connection between the disease gene function and pathology remain poorly understood. Enzymatic small GTPases act as molecular switches, which control fundamental cellular processes and are often correlated with various human pathological conditions. Studies from other and our laboratories demonstrated that three conserved and poorly characterized ADP-ribosylation factor-like (ARL) small GTPases, ARL3, ARL6, and ARL13B, act as prominent ciliary switches, with disrupted function predisposing human or mice to ciliopathies. The paramount obstacle being that the guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) that switch ciliary ARLs on and off, respectively, and the effectors of ARLs have not been identified. In other disorders, such as tuberous sclerosis, identifying the GTPase inhibited by the TSC2 GAP was transformative in understanding the disease, and we propose that the corresponding knowledge here would have a similar dramatic effect on understanding ciliopathies. Due to highly conserved cilia pathways and ciliopathy genes, Caenorhabditis elegans has been established as a simple and effective model for characterizing the physiological roles of ciliopathy proteins in their native cellular environments. In last fundig period, we have successfully established C. elegans as a model to investigate the roles of ciliopathy ARLs. Our recent data suggested that ciliary ARLs are likely organized into two distinct functional modules in the enigmatic inversin (InV) compartment of cilia. One function module contains ARL-13-ARL-3-NPHP-2-UNC-119, in which UNC-119 and nephronophthisis protein NPHP-2 act synergistically with ARL-13, but antagonistically with ARL-3, in regulating ciliogenesis. The second one contains ARL-6-ARL-13-BBSome, which may regulate cilia signaling through regulating the proper localization of ciliary sensory receptors. Our preliminary results also supported that the roles of ciliopathy ARLs are highly conserved from worm to mammalian cells. Based on these, our central hypothesis is that the three ciliopathy ARLs and their regulators are organized into distinct complexes to coordinate cilia biogenesis and signaling, respectively. We will employ C. elegans to identify in vivo regulators and functions, and mammalian systems to determine the applicability to human ciliopathies. Specific Aim 1 is to characterized the type of regulators for each component in ARL-containing protein module, and we hope to identify GEFs, GAPs, or effectors for ciliopathy ARLs; Specific Aim 2 is to ascertain whether and how ARL-13, NPHP-2, and UNC-119 coordinate IFT integrity and/or axonemal stability in the InV compartment, and whether ARL-3 antagonizes the roles of ARL-13-NPHP-22-UNC-119 through deacetylase HDAC-6-dependent manner; Specific Aim 3 is to determine whether the ARL-6-ARL-13-BBSome module coordinates cilia signaling through the mechanism that ARL-13 promotes ARL-6 activation, BBSome-cargo assembly, and subsequent proper ciliary localization of sensory receptors in the InV compartment. The proposed studies have great potentials for unveiling breakthroughs in cilia biology, and would provide seminal information about how cilia biogenesis and sensory function are regulated in their native environment, shed light on the etiologies of ciliopathies, and potentially provide novel targets fo disease diagnosis and treatment.